2.2 | Conjugation and release of cargo proteins
To demonstrate the conjugation and reductive release of cargo proteins, a model enzyme, namely β-galactosidase (β-Gal), was conjugated with lipid-coated PFH droplets. After cleavage of the disulfide bond under a strong reductive condition and successive centrifugation, the amount of β-Gal released into the supernatant was detected by measuring the enzymatic activity with a fluorogenic substrate, namely fluorescein-di-(β-D-galactopyranoside) (FDG). The enzymatic activity of the supernatants increased in response to the composition ratio of DPPE-PEG-PyDTP in the lipid coating of the droplets (see Supporting Information, Figure S3). This result suggested that β-Gal was conjugated with the droplets via disulfide bond formation with DPPE-PEG-PyDTP. Furthermore, the reductive release of β-Gal from the droplet surface was induced upon treatment with reduced glutathione (GSH), which results in a bio-reductive environment inside living cells. Reductive release of β-Gal was enhanced when using one to ten millimolar concentration of GSH (Figure 2A). A similar dependency on GSH concentration has been reported for conventional bio-reductive molecular tools that work in living cells[33]. From these results, the present conjugation method using DPPE-PEG-PyDTP was confirmed to be potentially suitable for intracellular protein release.